Vascular reactivity in sepsis and platelet dysfunction in septic shock.

Sepsis remains an important global cause of morbidity and mortality. Sepsis can be complicated by pathological vasodilation causing cardiovascular septic shock. The present study identifies that dysfunction of the RhoA/Rho-kinase (ROK) signalling pathway in vascular smooth muscle cells contributes to vasomotor dysfunction in sepsis. ROK inhibits myosin light chain phosphatase (MLCP) through Thr855 phosphorylation of MYPT, the L30 kDa myosin binding regulatory subunit of MLCP. MLCP dephosphorylates myosin light chain (LC₂₀) inhibiting the acto-myosin cross-bridge cycling underpinning vasoconstriction or platelet contraction. ROK dependent MLCP inhibition therefore favours vasoconstriction and can be indexed by Thr855-MYPT phosphorylation. Western blot analysis identified that Thr855 phosphorylation of MYPT was reduced in arterial segments isolated from a murine caecal ligation and puncture model of sepsis. Wire myography yielded data consistent with reduced contractile responses to thromboxane A₂ receptor stimulation, high [K⁺] mediated depolarisation and direct PKC stimulation. α₁₋ adrenergic receptor mediated vasoconstriction was similar in septic and non-septic animals, possibly reflecting the multiple mechanisms by which α₁₋ adrenergic agonists elicit vasoconstriction. Certain bacterial toxins and inflammatory mediators have the potential to attenuate ROK signaling; our data suggest therapeutic benefit of agents that promote MLCP inhibition or which vasoconstrict independent of the RhoA/ROK pathway. Current vasopressor strategies for septic shock primarily rely upon catecholamine therapy. However, there is interest in administration of vasopressin, an endogenous vasopressor inappropriately suppressed in septic shock. It is proposed that vasopressin mediates Ca²⁺ sensitisation through ROK mediated inhibition of MLCP, however, neither vasopressin dependent Ca²⁺ sensitisation nor Thr855 MYPT phosphorylation have been directly identified. In permeabilised rat caudal artery Ca²⁺ sensitisation was observed and found to depend at least partly upon PKC signaling. In contrast, stimulation with arginine vasopressin (AVP) was not associated with Thr855 MYPT phosphorylation despite the ROK inhibitor Y27632 attenuating vasopressin dependent vasoconstriction. These data support clinical evaluation of vasopressin therapy targeted to cases of septic shock arising from organisms capable of producing toxins, which neutralise RhoA/ROK. Furthermore, the data suggest either an MLCP independent vasoconstrictor role for ROK or ROK independent action of Y27632. Sepsis is also complicated by coagulopathy promoting both thrombosis and haemorrhage. Data regarding platelet function in sepsis is equivocal and absent in the specific subset of patients with septic shock. Recognising the importance of platelet contraction in thrombus formation and suggested similarities between vascular smooth muscle and platelet contraction we aimed to identify whether platelet contractile dysfunction contributed to impaired platelet aggregation in septic shock. Whole blood impedance aggregation was impaired in patients suffering from septic shock; deficits in aggregation correlated with illness severity. Impaired platelet aggregation was not associated with biochemical evidence of contractile dysfunction: neither Ser19-LC₂₀ nor Thr855-MYPT phosphorylation differed between septic shock and non-septic patients. These data indicate that therapeutic strategies to restore platelet function in septic shock might more profitably focus on platelet adhesion and secretion. These studies identify MLCP inhibition as a potential therapeutic avenue to ameliorate vascular smooth muscle, but not platelet, function in septic shock. Vasopressin might provide particularly effective vasoconstriction when targeted to cases of septic shock associated with disrupted ROK/MLCP integrity.Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 201

Calcium desensitisation in late polymicrobial sepsis is associated with loss of vasopressor sensitivity in a murine model

BACKGROUND: Sepsis is characterised by diminished vasopressor responsiveness. Vasoconstriction depends upon a balance: Ca(2+)-dependent myosin light-chain kinase promotes and Ca(2+)-independent myosin light-chain phosphatase (MLCP) opposes vascular smooth muscle contraction. The enzyme Rho kinase (ROK) inhibits MLCP, favouring vasoconstriction. We tested the hypothesis that ROK-dependent MLCP inhibition was attenuated in late sepsis and associated with reduced contractile responses to certain vasopressor agents. METHODS: This is a prospective, controlled animal study. Sixteen-week-old C57/BL6 mice received laparotomy or laparotomy with caecal ligation and puncture (CLP). Antibiotics, fluids and analgesia were provided before sacrifice on day 5. Vasoconstriction of the femoral arteries to a range of stimuli was assessed using myography: (i) depolarisation with 87 mM K(+) assessed voltage-gated Ca(2+) channels (L-type, Cav1.2 Ca(2+) channels (LTCC)), (ii) thromboxane A2 receptor activation assessed the activation state of the LTCC and ROK/MLCP axis, (iii) direct PKC activation (phorbol-dibutyrate (PDBu), 5 μM) assessed the PKC/CPI-17 axis independent of Ca(2+) entry and (iv) α1-adrenoceptor stimulation with phenylephrine (10(-8) to 10(-4) M) and noradrenaline (10(-8) to 10(-4) M) assessed the sum of these pathways plus the role of the sarcoplasmic reticulum (SR). ROK-dependent MLCP activity was indexed by Western blot analysis of P[Thr855]MYPT. Parametric and non-parametric data were analysed using unpaired Student's t-tests and Mann-Whitney tests, respectively. RESULTS: ROK-dependent inhibition of MLCP activity was attenuated in both unstimulated (n = 6 to 7) and stimulated (n = 8 to 12) vessels from mice that had undergone CLP (p < 0.05). Vessels from CLP mice demonstrated reduced vasoconstriction to K(+), thromboxane A2 receptor activation and PKC activation (n = 8 to 13; p < 0.05). α1-adrenergic responses were unchanged (n = 7 to 12). CONCLUSIONS: In a murine model of sepsis, ROK-dependent inhibition of MLCP activity in vessels from septic mice was reduced. Responses to K(+) depolarisation, thromboxane A2 receptor activation and PKC activation were diminished in vitro whilst α1-adrenergic responses remained intact. Inhibiting MLCP may present a novel therapeutic target to manage sepsis-induced vascular dysfunction.Benjamin AJ Reddi, John F Beltrame, Richard L Young, and David P Wilso

Review of Indications for Endotracheal Intubation in Burn Patients with Suspected Inhalational Injury

Inhalation injury is a major contributor to mortality following burn injury. Despite recognised clinical criteria to guide the intubation of burn patients, concerns remain regarding overutilisation of intubation. Complications can arise as a result of intubation, including ventilator-associated pneumonia (VAP). This study reviews the indications for intubation against the internationally accepted criteria (American Burns Association (ABA) and Denver criteria) for burn patients treated at the Royal Adelaide Hospital (RAH) burns unit between 2017 and 2020. Burn patients who were intubated on arrival to the RAH or in a pre-hospital setting were identified using the BRANZ database. Indications for intubation were compared to the ABA and Denver criteria. A total of 61 patients were identified with a mean total body surface area of 17.8%. A total of 95% of patients met one of the ABA and Denver criteria. The most common ABA and Denver criteria for intubation was deep facial burns or singed facial hair, respectively. Most adult patients with burns admitted to the RAH are intubated per published criteria. Early nasoendoscopy/bronchoscopy may be useful in determining patients who can be safely extubated within 48 h

High rate of persistent symptoms up to 4 months after community and hospital-managed SARS-CoV-2 infection

Recovery after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains uncertain. A considerable proportion of patients experience persistent symptoms after SARS-CoV-2 infection which impacts health-related quality of life and physical function

SARS-CoV-2 infects the human kidney and drives fibrosis in kidney organoids

This work was supported by grants of the German Research Foundation (DFG: KR 4073/11-1; SFBTRR219, 322900939; and CRU344, 428857858, and CRU5011 InteraKD 445703531), a grant of the European Research Council (ERC-StG 677448), the Federal Ministry of Research and Education (BMBF NUM-COVID19, Organo-Strat 01KX2021), the Dutch Kidney Foundation (DKF) TASK FORCE consortium (CP1805), the Else Kroener Fresenius Foundation (2017_A144), and the ERA-CVD MENDAGE consortium (BMBF 01KL1907) all to R.K.; DFG (CRU 344, Z to I.G.C and CRU344 P2 to R.K.S.); and the BMBF eMed Consortium Fibromap (to V.G.P, R.K., R.K.S., and I.G.C.). R.K.S received support from the KWF Kankerbestrijding (11031/2017–1, Bas Mulder Award) and a grant by the ERC (deFiber; ERC-StG 757339). J.J. is supported by the Netherlands Organisation for Scientific Research (NWO Veni grant no: 091 501 61 81 01 36) and the DKF (grant no. 19OK005). B.S. is supported by the DKF (grant: 14A3D104) and the NWO (VIDI grant: 016.156.363). R.P.V.R. and G.J.O. are supported by the NWO VICI (grant: 16.VICI.170.090). P.B. is supported by the BMBF (DEFEAT PANDEMIcs, 01KX2021), the Federal Ministry of Health (German Registry for COVID-19 Autopsies-DeRegCOVID, www.DeRegCOVID.ukaachen.de; ZMVI1-2520COR201), and the German Research Foundation (DFG; SFB/TRR219 Project-IDs 322900939 and 454024652). S.D. received DFG support (DJ100/1-1) as well as support from VGP and TBH (SFB1192). M.d.B,R.R., N.S., and A.A. are supported by an ERC Advanced Investigator grant (H2020-ERC-2017-ADV-788982-COLMIN) to N.S. A.A. is supported by the NWO (VI.Veni.192.094). We thank Saskia de Wildt, Jeanne Pertijs (Radboudumc, Department of Pharmacology), and Robert M. Verdijk (Erasmus Medical Center, Department of Pathology) for providing tissue controls (Erasmus MC Tissue Bank) and Christian Drosten (Charite´ Universitatsmedizin Berlin, Institute of € Virology) and Bart Haagmans (Erasmus Medical Center, Rotterdam) for providing the SARS-CoV-2 isolate. We thank Kioa L. Wijnsma (Department of Pediatric Nephrology, Radboud Institute for Molecular Life Sciences, Amalia Children’s Hospital, Radboud University Medical Center) for support with statistical analysis regarding the COVID-19 patient cohort.Peer reviewedPublisher PD